JPWO2020193483A5 - - Google Patents

Claims (20)

A method for isolating biomolecules from a cell culture, comprising:
(a) providing magnetic particles comprising ligands capable of binding to said biomolecules;
(b) contacting the cell culture containing the biomolecules with the magnetic particles to obtain magnetic particles containing the bound biomolecules;
(c) retaining the magnetic particles within a magnetic separator using a magnetic field;
(d) optionally washing the magnetic particles with a washing liquid;
(e) agitating the magnetic particles on at least one side of the magnetic separator to form a fluidized bed of magnetic particles within the magnetic separator;
(f) providing a flow of eluate substantially perpendicular to the at least one surface while retaining the magnetic particles within the magnetic separator using the magnetic field to remove the bound biomolecules; and eluting from said magnetic particles. 2. The method of claim 1, wherein step (e) comprises agitating the magnetic particles by varying the strength of the magnetic field, such as by applying an oscillating magnetic field. Step (b) or step (c) comprises adding the magnetic particles to a magnetic separator comprising at least one agitator, such as a plurality of agitators, and step (e ) comprises switching the agitator 3. The method of any one of claims 1-2 , comprising agitating the magnetic particles by turning on the . step (d) comprises the following substeps:
(d1) agitating the magnetic particles on at least one side to form a fluidized bed of magnetic particles;
(d2) dislodging the cell culture by providing a flow of wash fluid in a direction substantially perpendicular to the at least one surface while holding the magnetic particles within the magnetic field. 4. The method according to any one of 1 to 3 . Step (e ) includes agitating the magnetic particles to form a plurality of fluidized beds of magnetic particles in a plurality of substantially parallel planes of the magnetic separator, and step (f ) includes a plurality of 5. A method according to any one of claims 1 to 4 , comprising providing the flow of said effluent in a direction substantially perpendicular to a plane. an eluate in which said biomolecule is up to 10 bed volumes, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1 bed volume, preferably up to 4 bed volumes, more preferably up to 3 bed volumes 6. The method of any one of claims 1-5 , wherein the elution is performed using from claim 1, wherein at least steps (c) to (f 2 ), such as steps (b) to (f 2 ), etc. , are performed in said magnetic separator, preferably said magnetic separator is a high gradient magnetic separation system. 7. The method of any one of 6 . step (b)
(i) adding said magnetic particles to a magnetic separator, followed by providing said cell culture-derived feed to said magnetic separator, or (ii) said cell culture comprising said bound biomolecules. and said magnetic particles to a magnetic separator. 9. The method of any one of claims 1-8 , wherein steps (b) through (f) are performed within an integrated bioreactor vessel/contactor and magnetic separator. step (d) comprises the following substeps:
(i) removing the magnetic field; (ii) resuspending the magnetic particles;
(iii) contacting the magnetic particles with a portion of a wash solution;
(iv) holding the magnetic particles using a magnetic field;
(v) removing the washing liquid from the retained magnetic particles . 11. The method of any one of claims 1-10 , wherein step (d) is repeated at least once before proceeding to step (e) . Any of claims 1 to 11 , wherein the linear flow velocity of the eluate in step (f) or (e1) is in the range of 10-3000 cm/h, preferably in the range of 50-600 cm/h in step (f). The method according to item 1. Process according to any one of claims 3 to 12 , wherein the stirrer speed in step (e 1 ) is in the range of 15-1500 rpm, preferably in the range of 50-300 rpm. 14. The method of any one of claims 1-13 , wherein the magnetic separator is connected to a chromatography system. A method according to any one of the preceding claims, wherein said magnetic particles have a volume-weighted median diameter (d50, v) in the range 8-300 µm, preferably in the range 37-100 µm. A method according to any one of the preceding claims, wherein the magnetic particles have an average density of 1.05-1.20 g/ml of sedimented particles. 17. A method according to any preceding claim, wherein each of said magnetic particles comprises a porous polymer matrix and one or more magnetic granules embedded in said porous polymer matrix. 18. The method of claim 17 , wherein each of said magnetic particles comprises 5-15% by weight of said magnetic granules. A method according to claim 17 or 18 , wherein said magnetic granules have a volume-weighted median diameter (d50, v) of 1-5 µm. Each of said magnetic particles comprises a concentration of magnetic granules in a central region of the particle that is at least 200% of the concentration in a surface region of said particle, said central region being a distance greater than 0.2 times the particle radius from the particle surface. and the surface area is defined as having a distance from the particle surface of less than 0.2 times the particle radius.